The epidermal growth-factor receptor tyrosine kinase inhibitors have been effective in non-small cell lung cancer patients. However, acquired resistance eventually develops in most patients despite an initial positive response. Emerging evidence suggests that there is a molecular connection between acquired resistance and the epithelial–mesenchymal transition (EMT). N-cadherin is involved in the EMT and in the metastasis of cancer cells. Here, we analyzed N-cadherin expression and function in erlotinib-resistant lung cancer cell lines.H1650 cell lines were used to establish the subline resistant to erlotinib(H1650ER). Then, induction of the EMT was analyzed using immunostaining and western blots in H1650ER cells. N-cadherin expression in the resistant cells was examined using FACS and western blot. In addition, an invasion assay was performed to characterize the resistant cells. The effects of N-cadherin on cell proliferation and invasion were analyzed. The association of N-cadherin expression with the EMT phenotype was investigated using immunohistochemical analysis of 13 archived, lung adenocarcinoma tissues, before and after treatment with erlotinib.in vitro.In H1650ER cells, N-cadherin expression was upregulated, paralleled by the reduced expression of E-cadherin. The marked histological change and the development of a spindle-like morphology suggest that H1650ER cells underwent an EMT, accompanied by a decrease in E-cadherin and an increase in vimentin. A change in the EMT status between pre-and post-treatment was observed in 11 out of 13 cases (79%). In biopsies of resistant cancers, N-cadherin expression was increased in 10 out of 13 cases. Induction of the EMT was consistent with aggressive characteristics. Inhibition of N-cadherin expression by siRNA was tested to reduce proliferation and invasion of H1650ER cells Our data provide evidence that induction of the EMT contributes to the acquired resistance to EGFR-TKIs in lung cancer. It suggests that N-cadherin is a potential molecular target in the treatment of NSCLC. Lung cancer is the most common cause of death due to malignant carcinomas, and the use of traditional chemotherapeutic drugs against lung cancers are only modestly effective N-cadherin, a mesenchymal cadherin associated with the EMT, is crucial in cancer progression, with respect to both metastasis and to chemotherapy resistance. E-cadherin and N-cadherin share many structural and functional features. Both molecules establish calcium-dependent homophilic cell-cell adhesion with their extracellular domains and are connected with catenins at their intracellular domains This study evaluated whether induction of the EMT was related to the acquired resistance observed during erlotinib treatment and to determine the role of N-cadherin in those patients with NSCLC before and after erlotinib treatment.The human lung adenocarcinoma cell line H1650 was obtained from the Chinese Academy of Science  and was cultured in a humidified 5% CO2 incubator at 37°C, in a DMEM solution containing 10% FBS, penicillin and streptomycin. The erlotinib-resistant cell line (H1650ER) was established by exposing parental cells to increasing concentrations of erlotinib, up to 10 µM, for several months. The resistant cells were continuously maintained in a medium containing 10 µM of erlotinib prior to each experiment.Mouse anti–E-cadherin antibody(sc-21791) and monoclonal anti-vimentin antibody(sc-53464), Mouse anti–β-actin antibody(sc-47778), Rabbit anti–N-cadherin antibody (sc-59987), fluorescein isothiocyanate-conjugated goat anti-mouse antibody(sc-53800), and PE-conjugated goat anti-mouse antibody (sc-53464) were obtained from Santa Cruz Biotechnology . Akt(#9272), S473 p-Akt(#4060), EGFR(#2239), and p-42/44 MAPK(#4695) were all purchased from Cell Signaling Technology . EGFR [pY1068] (CB11007889) and Rabbit anti–Erk antibody(CB1241938) were purchased from Affinity BioReagents .4/ml, were incubated with different concentrations of erlotinib in 96-well culture plates for 48 hours. Next, the culture medium in the 96-wells was replaced with fresh medium containing 5% CCK-8 reagent; after an hour, the absorbance at 450 nm was measured and the values were corrected by subtracting the absorbance of control wells that did not contain cells.Cell proliferation assays were measured using the Cell Counting Kit-8 assay , which uses the bioreduction of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H tetrazolium sodium (WST-8) to orange-colored formazan to measure cell viability. Briefly, 100 µl of H1650 and H1650ER cells, at 2×10Tissues from 13 cases of lung adenocarcinoma, from both before and after erlotinib treatment, were collected between January 2008 and July 2011 in Henan Provincal People’s Hospital, and embedded in paraffin. Tumor tissue slides were de-paraffinized and dehydrated using Slide Brite . Antigen retrieval was performed using 0.05 M Glycine-HCL buffer, pH 3.5, containing 0.01% (w/v) EDTA, at 95°C for 20 min and stained with an antibody against human E-cadherin, vimentin and N-cadherin, which are same as for the in vitro assays,. The evaluation of immunostaining of these sections was made blind to two trained pathologists who were unaware of the clinical background of the samples. The intensity of E-cadherin,vimentin and N-cadherin were scored and placed into four categories according to staining: 0 for 0%; 1 for 1–33%; 2 for 34–66%; and 3 for 67–100%. All of the 13 human tissues involved in our study were carried out according to the principles set out in the Declaration of Helsinki 1964 and all subsequent revisions. The study was approved by the Ethics Committee of Henan provincial people’s hospital, Written informed consent was obtained from all subjects.Small interfering RNA (siRNA) specific for N-cadherin was purchased from Invitrogen. The sequence used for the N-cadherin interfering RNAs (siRNA) was 5′-CUAACAGGGAGUCAUAUGGUGGAGC-TdT-3′. To minimize nonspecific effects of interfering RNAs, non-targeting control siRNA, also purchased from Invitrogen, was used as negative control. The siRNA transfection reagent (Invitrogen) was used according to the manufacturer’s instructions. The experiment was repeated at least three times, independently.4 cells in 100 µl RPMI medium, with 1% FCS were seeded in the upper compartment, and 600 µl RPMI, with 10% FCS were added to the lower chamber. Cells were allowed to migrate for 24 hours at 37°C. After removing cells on the upper side of the transwell, cells on the underside were stained with 0.1% crystal violet solution (Becton Dickinson) and were lysed with 10% acetic acid for quantification by densitometric measurement at 550 nm. Experiments were carried out in triplicate.Cells were treated with N-cadherin-specific siRNAs for 48 hours to knockdown N-cadherin expression. Invasion was observed using transwell culture inserts with a 6.5 mm diameter and 8 µm pore filters . Then, 3×10Proteins from the cell lysates, which were prepared using RIPA buffer  supplemented with protease inhibitors , were detected by SDS-PAGE and then electrotransferred to Immobilon membranes (Invitrogen); these membranes were subsequently probed with specific antibodies. The secondary antibodies used horseradish peroxidase-conjugated antibodies and the membrane was developed using an ECL kit (Thermo). This experiment was repeated at least three times, independently.4 cells per well in 0.35% agarose in DMEM medium layered on top of 0.7% agarose in the same medium. Colony formation was observed after 2–3 weeks in culture. Following staining with 0.005% crystal violet, colonies were counted under a microscope. Experiments were carried out in triplicate. The number of cells were counted and averaged to determine the cloning efficiency.The H1650 and H1650ER cells were treated with 0.05% trypsin and plated in 6-well plates at 1×10Cells grown on coverslips were washed three times with PBS. They were fixed for 10 min with cold methanol and washed three times with PBS. The coverslips were then blocked in a solution of PBS, 10% bovine serum, and 0.1% Tween 20 for 1 h. The cells were incubated with primary antibody to either E-cadherin or to vimentin at 2 µg/ml for 1 h, then the cells were washed twice with PBS, and incubated with secondary antibodies diluted in PBS, 5 or 10% bovine serum, and 0.1% Tween 20. After incubating on coverslips for 1 h at 37°C the coverslips were washed as described above and mounted on slides in medium containing DAPI.5 cells in PBS were placed in 96-well V-bottom plates and incubated at RT for 1 h with IgG. Cells were washed twice with PBS. Following the washes with PBS, bound antibody was detected using goat anti-Human-PE or goat anti-Human-CY3, and analyzed by FACS .Cancer cells were dissociated using 0.05% trypsin; they were washed with PBS and 100 µl aliquots of 10The SPSS 13.0 statistical package  was used for data analyses. The significance of the difference among the covariates was determined by two-tailed v2 test. P values less than 0.05 were considered statistically significant.The erlotinib-resistant lung cancer cell line H1650 (H1650ER), originally derived from the parental H1650 cell line, demonstrated an increased dependency on EGFR signaling for growth, as described previously Subsequently, we examined the mechanism of resistance to the EGFR-TKI erlotinib. Using Western blots, we probed lysates from both parental H1650 and H1650ER cells treated for 6 hours with erlotinib. Erlotinib treatment effectively blocked EGFR and Akt phosphorylation in the parental H1650 cells but not in the H1650ER cells. In these resistant cells, P-Akt was up-regulated and was evaluated as a candidate for involvement in the mechanism of acquired resistance. In contrast to the persistent Akt phosphorylation, Erk phosphorylation was down-regulated by erlotinib treatment in the resistant cells. These results suggested that the erlotinib-resistant cells adopted a new mechanism for activating the PI3K/Akt pathway .To identify markers of erlotinib resistance, we compared gene expression in paired parental and resistant cell lines. N-cadherin expression was highly elevated in H1650ER cell lines, which we confirmed by FACS and western blot. . Next, mTo identify the expression of N-cadherin and EMT biomarkers in EGFR-mutant NSCLC tissue samples, we performed biopsies on patients before erlotinib treatment and at the time that drug resistance was acquired. Thirteen patients had tumor tissue available both before and after TKI treatment. The characteristics of the 13 patients are shown in Of the total cases, 12 out of 13 cases were negative for N-cadherin with the exception of one adenocarcinoma case that had focal N-cadherin expression before erlotinib treatment. On the other hand, the expression of N-cadherin was increased in 10 out of 13 biopsies in resistant cancers. Immunohistochemical expression were summarized in Induction of the EMT was consistent with aggressive characteristics, such as increased clonogenic growth, cell motility and invasion. To further characterize H1650ER cells, we compared the growth of parental and resistant cell lines using a cell-counting assay; the percentage of viable cells was calculated relative to the untreated controls . We alsoThe H1650ER cells expressing high levels of N-cadherin were down modulated by transient transfection with siRNAs. These efficient siRNAs produced significant reductions in N-cadherin protein  when comHere, we present evidence showing that acquired resistance to, EGFR-tyrosine kinase inhibitor, erlotinib in lung cancer is mediated by the epithelial–mesenchymal transition (EMT). In this study, we found that H1650 lung cancer cells developed resistance to erlotinib and underwent EMT phenotypic changes, which were consistent with decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers vimentin. Moreover, in these clinical samples, the pretreatment cancers exhibited adenocarcinoma histology with preserved membranous staining for E-cadherin, while positive expression for vimentin was more frequently recognized in advanced, posttreatment cancers, indicating that some tumors underwent the EMT. Notably, our data indicate that EMT is associated with erlotinib resistance in NSCLC, as previously reported in vitro. The aberrant N-cadherin expressive status both in vitro and in vivo illuminates our hypothesis that, the crucial roles of N-cadherin reside not only in EMT, but also in tumor metastasis and erlotinib resistance. However, more expremental data are in need to verify our hypothesis. As not all the lung adenocarcinoma are sentitve to TKI, as only a few cell lines are sensitive to TKI, such as A549, PC9, HCC827, CALU-3, HCC4006 those harboring the acquired mutation in the EGFR gene Aberrant expression of cadherins, or cadherin switching, is one of the key events in the EMT in certain types of cancer Other researchers have reported that N-cadherin controls the motility and migration of cancer cells by suppressing Akt phosphorylation In summary, our study provides a further insight into the mechanisms involved in NSCLC and TKI resistance, revealing that the upregulation of N-cadherin in H1650 ER cells leads to increased tumor cell migration, invasion and tumorigenic potential. Our results also suggest that the maintenance of the EMT phenotype in H1650ER cells may be related to the sustained expression of N-cadherin. Therefore, N-cadherin may serve as a promising new target for the treatment of cancers with acquired resistance to EGFR TKIs. Because N-cadherin is expressed on the cell surface, we also ponder whether therapeutic targets using N-cadherin–specific monoclonal antibodies would have efficacy in those cancer cells with acquired resistance to EGFR tyrosine kinase.


The ETT provides a conduit for bacterial access to the lower respiratory tract and a substratum for biofilm formation, both of which lead to VAP. A novel microscopic ordered surface topography, the Sharklet micro-pattern, has been shown to decrease surface attachment of numerous microorganisms, and may provide an alternative strategy for VAP prevention if included on the surface of an ETT. To evaluate the feasibility of this micro-pattern for this application, the microbial range of performance was investigated in addition to biofilm studies with and without a mucin-rich medium to simulate the tracheal environment Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter baumannii, and Escherichia coli, were evaluated for attachment to micro-patterned and un-patterned silicone surfaces in a short-term colonization assay. Two key pathogens, MRSA and Pseudomonas aeruginosa, were evaluated for biofilm formation in a nutrient rich broth for four days and minimal media for 24 hours, respectively, on each surface type. P. aeruginosa was further evaluated for biofilm formation on each surface type in a mucin-modified medium mimicking tracheal mucosal secretions. Results are reported as percent reductions and significance is based on t-tests and ANOVA models of log reductions. All experiments were replicated at least three times.The top five pathogens associated with ETT-related pneumonia, Methicillin-Resistant p < 0.05) reduction compared to un-patterned controls. Biofilm formation was also reduced, with 67% (p = 0.12) and 52% (p = 0.05) reductions in MRSA and P. aeruginosa biofilm formation, respectively. Further, a 58% (p < 0.01) reduction was demonstrated on micro-patterned surfaces for P. aeruginosa biofilms under clinically-simulated conditions when compared to un-patterned controls.Micro-patterned surfaces demonstrated reductions in microbial colonization for a broad range of species, with up to 99.9% (in vitro. Future application of this micro-pattern on endotracheal tubes may prevent or prolong the onset of VAP without the need for antimicrobial agents.This engineered micro-pattern reduces the colonization and biofilm formation of key VAP-associated pathogens  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which are largely responsible for HAIs and are the primary organisms demonstrating antimicrobial resistance, thereby escaping the effects of antibacterial drugs [S. aureus, P. aeruginosa, K. pneumoniae, E. coli, and A. baumannii cause 28%, 22%, 10%, 7% and 7%, respectively, of first episode VAP and HAI-associated pneumonia infections world-wide making them the top five causative agents of these infections [S. aureus (MRSA) and P. aeruginosa comprise the top two causative organisms of VAP and are considered particularly devastating lung pathogens as they cause persistent pneumonia infections, are resistant to a number of antimicrobials, and are associated with a high attributable mortality of patients with VAP [Pneumonia is the most common hospital-acquired infection (HAI) in Intensive Care Units (ICUs)—comprising some 27% of these infections and driving more than half of the antibiotics prescriptions in the ICU ,2. Vential drugs ,7. CurreVAP is attributed to the endotracheal tube (ETT) used during ventilation ,2,5,8. Ie.g. clinical presentation, local drug resistance patterns, patient exposure) and this complexity prevents the standardization of a VAP antimicrobial treatment guideline [Treatments for VAP universally include the use of antimicrobials to clear the infection. Prescribing the appropriate antimicrobials at the onset of VAP is increasingly difficult but extremely important, as an inadequate initial antimicrobial therapy is associated with increased mortality ,18. Numeuideline ,20. Broauideline . It is auideline -24. CommTo prevent the overuse of anti-microbial treatments, device modifications and improved patient care practices have been implemented to reduce microbial access to the lower respiratory tract. The addition of a subglottal secretion port as well as reducing the thickness of the ETT cuff prevents microaspiration of subglottal secretions into the lungs. These device modifications, in addition to elevation of the patient’s head, daily sedation interruptions, and oral antiseptic treatments, have shown some benefit , but theE. coli and S. aureus biofilm formation in vitro without the use of antimicrobial agents [in vitro conditions.Engineered surface topographies, particularly geometries of ordered features designed with unique roughness properties, elicit specific, predictable biological responses and have been shown to control bio-adhesion -29. Studl agents ,32. The Micro-patterned-surface samples and un-patterned (control) surface samples were created as described previously . BrieflyP. aeruginosa, K. pneumoniae, A. baumannii and E. coli. Two additional strains of P. aeruginosa were also assessed to confirm the validity of the results across multiple strains within a species: a second clinical isolate and a laboratory strain. The laboratory P. aeruginosa strain PA14, carrying a ∆bifA mutation, was included in these studies as a comparative strain control due to its genetic modification to overproduce exopolysaccharide and thus form biofilms quickly and consistently [All clinical isolates analyzed in this study were purchased from ATCC Table . These sistently . A singl7 CFU/mL for incubation time of 1 to 4 hours statically at room temperature depending on the propensity of the species to colonize the surface; the cell density on un-patterned controls was targeted to remain within an average of 2.5 to 6 logs . Samples were immersed in inoculum containing ~106 CFU/mL in TSB. Samples were immersed with 20 mL of the bacterial suspension statically at 37°C for four days, with gentle media replenishing each day from the center of the dish. For P. aeruginosa biofilms: Overnight cultures of P. aeruginosa ATCC9027 and P. aeruginosa PA14ΔbifA were diluted into minimal M63 media supplemented with 0.4% arginine 2SO4, 1 mM MgSO4), or minimal M63 media supplemented with 0.4% arginine, 2 mg/mL porcine mucin that contains the MucA/B complex found in mucus (Sigma Aldrich), and 400 μg/mL oxacillin (Sigma Aldrich), to a final bacterial concentration of 106 CFU/mL [10 transformed before statistical analysis. All biofilm experiments were completed in triplicate.Micro-patterned and un-patterned control samples were suctioned to the bottom of a sterile Petri dish around the outer perimeter of the dish using ethanol. For MRSA biofilms: Overnight cultures of MRSA (ATCC700698) were diluted into fresh TSB to a final bacterial concentration of 106 CFU/mL ,35-37. S6 CFU/mL . Each bi10(micro-pattern data points) from the average log10(un-patterned control data points). After confirming the normality of the log reductions by residual and normal probability plots, the mean log reduction was interpreted as the median percent reduction with the equation: 1-10(− LR). Statistical significance of the reductions for the colonization and biofilm assays were assessed using a 1-sided t-test of log reductions from at least three experiments per organism. Estimates of the among- and within-experiment variances were assessed using ANOVA of the log transformed cell densities for each un-patterned control sample and micro-patterned sample, with a random effect for experiment. The repeatability standard deviation (SD) of the un-patterned sample, which quantifies the repeatability of the methods based on control variances, is the square root of the sum of the among-experiment variance and the within-experiment variance divided by the number of replicates per experiment [A log reduction (LR) per experiment was calculated by subtracting the average logperiment . All anaP. aeruginosa, K. pneumoniae, A. baumannii and E. coli. Two additional strains of P. aeruginosa were also evaluated to confirm the validity of the results across multiple strains within a species: a second clinical isolate and a laboratory, hyper-biofilm forming strain. The micro-patterned surface significantly reduced the colonization of all species and strains compared to un-patterned controls, with median reductions in CFU counts ranging from 95.6% to 99.9%, p < 0.05 when evaluated across multiple experiments  was calculated across experiments for each organism  median reduction of MRSA biofilm volumes grown over a four day duration in a TSB media when compared to un-patterned control surfaces  was observed ; GO is a biofilm expert and professor at Dartmouth Medical School and an un-paid collaborator with STI; AP is a statistician and professor for the Center of Biofilm Engineering at Montana State University and a consultant for STI; AB is a materials engineer and professor at the University of Florida, Gainesville and is the inventor of the micro-pattern technology where he continues to consult on the technology for STI.


Wolbachia to invade and persist in host populations. However, mechanisms that underlie the invasion and persistence of non-CI strains are less well understood. Here, we established a spider mite Tetranychus phaselus population multiply infected by Cardinium as well as two distinct lineages of Wolbachia, designated wCon and wOri, to study the forces driving the spread of the non-CI strain of Wolbachia wOri. Interestingly, we found that wOri provided a longevity advantage to its female hosts under ideal conditions, making wOri stay longer in this population, and then being transmitted to more offspring. Furthermore, the lifespan of uninfected females was reduced when mated with multiple-infected males. As a result, the uninfected population is attenuated by the multiple-infected males. Thus, we infer that the host age effects of multiple infection may represent sufficient forces driving the spread of wOri through the host population.Cytoplasmic incompatibility (CI) has been proposed as a major mechanism by which certain strains of  Wolbachia are obligate intracellular rickettsia-like bacteria that occur in numerous invertebrates. Infection with Wolbachia is found frequently in insects Cardinium from the Bacteroidetes group has been found in four insect orders and approximately 6–7% of arthropods species that have been examined Wolbachia to invade host populations Drosophila simulansAedes albopictusWolbachia to invade and persist in host populations, mechanisms that underlie the invasion and persistence of non-CI strains are less well understood. It is surprising, therefore, that spread mechanism of Wolbachia symbioses has not all been identified yet, especially in view of the fact that several Wolbachia strains have invaded natural populations even though they induce little to no CI WolbachiaSeveral reports have noted that CI, the most common of the reproductive abnormalities, imparts the ability to Tetranychus phaselus is a serious agricultural pest that damages many crops including cotton, bean, cucumber, eggplant, pepper, tomato, cucurbits, papaya, passion fruit. Infections of both Wolbachia and Cardinium in the same host are known in a number of species in the Acari and the Hymenoptera Wolbachia occurred commonly in several species, Drosophila simulansAedes albopictusNasonia vitripennisCallosobruchus chinensisDrosophila parasitoid Leptopilina heterotomaTetranychus cinnabarinus (T. urticae red form) T. phaselus. Wolbachia-superinfection (that is co-infection with two or more strains), and Cardinium-infection occur naturally in the same host. To our knowledge, this is the first case of multiple infection with Cardinium and two strains of Wolbachia in arthropods.The spider mite wOri is spread in co-existence with wCon and Cardinium in T. phaselus. To this end, we established three lines of T. phaselus, an uninfected line (Wolbachia-free and Cardinium-free), a double-infected line (infected by wCon and Cardinium), and a multiple-infected line .We examined the levels of CI induced in T. phaselus by double-infection and multiple-infection, and compared the longevity, egg hatch, fecundity and bacteria densities in the three lines of T. phaselus. Our results reveal that wOri has effects on host age that may act as new, non-CI forces for the spread of a Wolbachia strain.The aim of the present study was to discover the mechanism by which Tetranychus phaselus was collected from soybean [Glycine max (L.) Merr.] leaves in Cixi, Zhejiang Province, east China in July 2009. Mites were reared on a leaf of the common bean (Phaseolus vulgaris L.) placed on a water-saturated sponge mat in Petri dishes (dia. 9) at 25±1°C, 60% r.h. and under L16-D8 conditions.The spider mite Wolbachia and Cardinium infection status by PCR amplification. The eggs were separately reared on new leaf discs depending on the infection status of the mother. The above process was continued for three to four generations until a 100% infected population was obtained. The eggs of the uninfected mothers were reared to establish the uninfected line.In order to cross infected and uninfected individuals, 100% double-infected, 100% multiple-infected and 100% uninfected lines were prepared for the population. One female from the teleiochrysalis stage was allowed to lay eggs without being crossed with males. The eggs were reared until adulthood . After the males reached sexual maturity, they were backcrossed with the mother. Then, the female adults were transferred to new leaf discs and were allowed to lay eggs for 3–5 days. A female was checked for In order to exclude the influence of different body sizes of female and male mites, DNA was extracted by homogenizing a single adult mite in a 15 µl  or 25 µl  mixture of STE buffer  and proteinase K  in a 1.5 ml Eppendorf tube. The mixture was incubated at 37°C for 30 min and then 95°C for 5 min. The least bacteria DNA is lost with this method of DNA extraction.Wolbachia and Cardinium infection, all PCR reactions were run in 25 µl buffer using the TAKARA Taq kit : 16.3 µl H2O, 2.5 µl 10×buffer, 1.5 µl of 2.5 mM deoxyribonucleotide triphosephates (dNTPs), 1.5 µl of 25 mM MgCl2, 0.2 µl Taq polymerase , 2 µl sample and 1 µl primers (10 mM each). The primers used for the detection of Cardinium were CLOf and CLOr Wolbachia infection status of 38 Chinese populations of agriculture mites, including T. kanzawai, T. cinnabarinus, T. urticae, T. phaselus and Amphitetranychus viennensis. The results suggest that all Wolbachia infected in Chinese populations of agricultural mites have been classified into subgroup Ori and Con belonging to supergroup B, according to the wsp gene sequences. In order to monitor the Wolbachia strains in those agriculture mites, we designed specific primers, wCon –F (5′-AAATGCAACAGGTAAAGAAAAGG-3′) and wCon-R (5′-CAAATCCTTTTTGATCTTTAACTGTA-3′), wOri –F (5′-ACATATAAATCAGGTAAGGACAACA-3′) and wOri –R (5′-CCAGATTTTTTATCACCAGTAACC-3′). Both of the Wolbachia-specific wsp primers were designed by Primer5 according to the wsp sequence of wOri and wCon, which were deposited in GenBank , which amplified ca. 320 bp of wsp gene. Cycling conditions were 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s and a final extension of 5 min at 72°C.To check for Wolbachia and Cardinium-specific primers, primers par COI-forward and COI-reverse wOri, wCon and Cardinium.For samples failing to amplify using Wolbachia strains and Cardinium influenced CI modification and rescue, crossing experiments were performed  and mortality of offspring. Fecundity was estimated as the total number of eggs laid in the first 5 days. Data were analyzed with one-way analysis of variance (ANOVA), and the means were compared using the Tukey-HSD test (SPSS 17.0). To normalize the data, log transformation was used for the number of eggs laid per female, and arcsine square root transformation was used for egg hatchability, sex ratio and mortality.To reveal how the different erformed . Female wOri, wCon and Cardinium in individual mites were estimated by Quantitative PCR. Quantitative PCR was carried out with the ABI PRISM 7300 Sequence Detection System (Applied Biosystems). The 20 µl reaction mixture consisted of 10 ul 2×SYBRq Premix Ex Taq (Applied Biosystems), 0.4 µl 10 mM of each primer, 0.4 µl 50×ROX Reference Dye, 2 µl DNA template and 6.8 µl H2O in single wells of a 96-well plate (PE Applied Biosystems).Bacteria densities were measured in the first 20 days of the life span of females and in the first 8 days of the life span of males (because of their shorter longevity and the higher escape frequency). Copy numbers of Cardinium 16S rDNA gene (133 bp) and Wolbachia wsp gene , the following primers were designed and used: wOriQ-F (5′-GCA GCG TAT GTA AGC AAT CC-3′), wOriQ-R (5′-ATA ACG AGC ACC AGC ATA AAG-3′); wConQ-F (5′-CTC GTT ACT TCG GTT CTT ATG GC-3′), wConQ-R (5′-TTA AAC GCT ACT CCA GCT TCT GC-3′); cardQ-F (5′-CCT GGG CTA GAA TGT ATT TTG-3′), cardQ-R (5′-AAA GGG TTT CGC TCG TTA TAG-3′). PCR primers and probes were designed using Primer 5. The Q-PCR cycling conditions included 1 cycle (10 s 95°C) followed by 40 cycles , and finally 1 cycle . Eight super-infected males of 1-, 4-, 8- and 12-day-old were collected. DNA of single mites was extracted using the above method. Three replicates were run and averaged for each DNA sample. Negative controls were included in all amplification reactions.For the selective amplification of a small portion of the −7 to 10−3 dilutions of the DNA standards prepared from plasmid DNA. The quality and concentration of all purified standard DNA were measured by OD absorbance at 260 nm. The number of molecules in all samples was determined from the threshold cycles in the PCR based on the standard curve. Statistical analysis was performed using the Mann-Whitney U-test.Standard curves were plotted using a 10-fold dilution series consisting of 10Differences in host longevity were observed in comparisons of the different infection types. We measured age-specific survival of the females that are uninfected, single-infected and double-infected. For the infected females, we compared the longevity difference between single-infected and double-infected virgins. Meanwhile, the longevity of uninfected females was also measured. They are divided into four groups, according to their different mating patterns, including virgins as well as those mated with uninfected, single-infected and double-infected males. In order to test the longevity of virgins, 50 teleiochrysalids  were placed on the different leaves according to the infection patterns. The new adults emerging within 24 hours were tested for longevity. For the longevity of mated females, every newly emerging adult was separately placed with two males with different infection statuses  on the same leaf disk. The males were allowed to stay with females for 48 hours, which was designed to avoid the variance in mating times. Leaves were monitored every day, and dead females were removed and counted until all females had died. Survivor curves for individual hosts were compared using the Kaplan-Meier log-rank test (SPSS 17.0).Wolbachia infection, among which four were infected only by wCon and nine were superinfected by both wOri and wCon. Moreover, we detected Cardinium 16S rDNA in all theWolbachia positive mites. However, we did not discover any individuals singly infected with Cardinium, wOri or wCon. As a result, we established 3 lines including a 100% uninfected line (U), a 100% double-infected line  and a 100% multiple-infected line .Thirteen out of forty mites in a rearing group were positive in PCR assay for In order to clarify the effect of wOri on host reproduction, four crosses between Iwc and Iwwc were conducted.wOri was investigated (wOri (Iwwc) were not able to induce CI with single-infected females (Iwc). Therefore, under this intricate infection status, wOri existed as a non-CI strain in this spider mite population. Meanwhile, CI expression of double-infected (Iwc) and multiple-infected (Iwwc) males was also contrasted. There was no significant difference in hatchability and sex ratio between the two predicted CI crosses , indicating that wOri does neither contribute to induce a higher level of CI nor induce additive effects on CI expression.The hatchability of eggs was compared among the four crosses in which the CI of stigated . A, B. Nmultiple-infected males (♀U×♂Iwwc) was (23.84±4.63)%, significantly lower than that found in compatible crosses (93.2%–96.7%)  showed a strong reduction in egg hatchability and a strongly male-biased sex ratio. In the predicted incompatible cross (♀U×♂Iwc), on average, (26.02±3.94)% of all eggs hatched, against 93.5–93.8% in the other crosses .C. Simil%–96.7%) .E. The s%–96.7%) .D, F. ThwCon and Cardinium. (ii) The fecundity of multiple-infected females can be increased by mating with uninfected males, but decreased by mating with double-infected males. It means that double-infected males act as a block to expansion of multiple infection by weakening the fecundity of multiple-infected females. Based on this point, we agree with your opinion that there is a competition between the doubly and triply infected females. (iii) Both the double-infected and multiple-infected males strongly depress the fecundity of their uninfected mates. In particular, multiple-infected males caused an even greater depression of fecundity.Fecundity of uninfected, single-infected and super-infected females was compared by analyzing the number of eggs laid in the first 5 days (Table.1). From these crosses we can draw three conclusions: (i) Mating with uninfected males increases the fecundity of double-infected females, which consequently promotes the spread of wOri, wCon and Cardinium in the double- and multiple-infected individuals, in which the CI strength seemed to be related to the bacteria density. Our hunch proved to be correct: wOri was present at a rather low density in the multiple-infected males, being only 0.1%–0. 2% of the densities of wCon and Cardinium , infection with wOri enhanced the proliferation of wCon and Cardinium. Furthermore, the dynamic of wCon density during host aging are affected by wOri infection. wCon density increased more rapidly in multiple-infected females (from (0.40±0.04)×108/ml to (4.95±0.14) ×108/ml) than in double-infected females (from (0.46±0.03)×108/ml to (3.87±0.39) ×108/ml) during the first 16 days of their life spans ×108/ml on the first day post eclosion (8 and (4.64±0.29)×108 copies per ml (P>0.05). However, in multiple-infected females, Cardinium density rose from (1.07±0.11)×108/ml to (7.10±0.46) ×108/ml step by step from the first day to the sixteenth day post eclosion, and then, it fells to (5.43±0.52)×108/ml on the twentieth day. Mann-Whitney tests indicated that the differences between each pair of two ages were significant (P<0.05).In order to reveal whether the existence of parately .The doubfe spans .a. In adeclosion .b. Howevs per ml . During wOri had on its female hosts, we measured the longevities of uninfected (U), double-infected (Iwc) and multiple-infected (Iwwc) females. Because of the variously uncertain factors in the environment, it is almost impossible to obtain the natural dynamics of the longevity of the hosts on different infection statuses. In order to exclude any influence of differences in environmental conditions, we measured the females on different infection statuses under ideal conditions, including no predation, no food limitation and no mating behavior. On ideal conditions , we found that wOri infection prolonged female survival . Consequently, wOri can remain longer in the female hosts, which allows it to be transmitted to more offspring. We regard this phenomenon as a force causing wOri to persist in this population under ideal conditions.To discover the effects that survival .a. This 2 = 0.696, df = 1, P = 0.404). Third, mating with multiple-infected males dramatically shortened the life span of uninfected females . The difference in longevity between females mated with uninfected males and females mated with multiple-infected males was significant . Also, females mated with multiple-infected males have a significantly shorter life span than females mated with double-infected males . These results clearly show that the longevity of uninfected females mated with multiple-infected males was further shortened by not only mating behavior, but also the spermatozoa modified with wOri. The ability of wOri to reduce the longevity of uninfected females by their spermatozoa may act as an additional force to drive the spread of wOri through a mite population. This effect of wOri gives extra strength to a multiple-infection to invade an uninfected population.To determine the effect of infection status of males on the longevity of females, we compared the longevities of different groups of uninfected females which are virgins, and those separately mated with uninfected, double-infected and multiple-infected males. From the survival curves of these four groups of females .b, threeCardinium and two strains of Wolbachia in arthropods. A possible mechanism of the multiple infections in the wild is horizontal transmission. An infection survey of wild spider mite populations in Zhejiang Province shows that Tetranychus truncatus, another important agriculture pest co-existing in this area, is singly infected by wOri (own unpublished data). Probably, wOri in T. phaselus was acquired through horizontal transmission from T. trunctatus. It could also be horizontal transmitted from T. phaselus to T. trunctatus. Another possibility is that both species could have inherited the triple infection from a common ancestor and Cardinium and wCon became lost in T. trunctatusTo our knowledge, this is the first case of multiple infection with wOri and the other bacteria (wCon and Cardinium) in the same host. wCon and Cardinium share their intensive CI with wOri, although wCon and Cardinium are dominant strains over wOri, which compensates for the CI absence of wOri. Even though wOri suppressed the densities of wCon and Cardinium in the four-day- old multiple-infected females because of the limited nutrients in the young hosts, it had favorable effects on the densities of wCon and Cardinium in the older hosts  the strength of CI, (ii) maternal transmission efficiency, and (iii) fitness effects on the host The powerful spreading capability of Wolbachia in spider mites in the laboratory wOri is perfect probably, ensuring its maintenance in the T. phaselus population. However, future experiments are needed to examine our conjecture.An empirical study directly examined the transmission efficiency of Wolbachia infections. The intensity of CI is an important parameter that determines the spread and maintenance of Wolbachia infection in the host population Wolbachia spread, as witnessed in nature in Drosophila simulansWolbachia-transinfected A. aegyptiWolbachia induce high CI, which leads to rapid population invasion. For instance, the Wolbachia strain wRi has been shown to spread geographically at a rate of 100 km/year into uninfected D. simulans populations through the action of CI Cytoplasmic incompatibility (CI) is most commonly associated with Wolbachia super-infections (that is co-infection with two or more Wolbachia strains) occur naturally wOri did not induce a higher level of CI, indicating that wOri did not have an additive effect. The bacteria density measurements lend qualified support to the hypothesis that wCon and Cardinium interfere with the ability of wOri to cause CI by suppressing wOri densities. wOri was present in the lowest copy number among those bacteria in males, being only about 0.1%–0.2% of the densities of wCon and Cardinium. Obviously, wCon and Cardinium are dominant species compared with wOri. Similarly, suppression of the density of one symbiont by the other has been found before in natural infections Wolbachia super-infection created in mosquitoes by Walker et alWolbachia with developing sperm show that an abundance of Wolbachia in the testes is required to induce CI. We hypothesized that the testes of T. phaselus have been occupied by wCon and Cardinium, leaving limited room for wOri. This is a potential mechanism of undetectable CI of wOri, which could be further proved by localizing the symbionts in testes of multiple-infected males wCon and Cardinium could not be investigated because of the absence of lines of singly infected with wCon or Cardinium.Wolbachia, such as wAu from D. simulans and wMel from D. melanogaster, and wStri in the brown planthopper Nilaparvata lugenswOri, because they have a greater number of offspring. In addition, the non-CI wOri appears to have a novel mechanism for invading and diffusing through a population. The survival curves of uninfected females indicate that the shorter life span of uninfected females induced by mating with multiple-infected males acts to spread wOri specifically in multiple-infected individuals. This is because the multiple-infected spermatozoa, which decrease the longevity of uninfected females, help to decrease the number of uninfected offspring. We conjecture that the ability of reduction of the longevity of uninfected females probably acts as a new and specific force for wOri to invade and spread through this spider mite population. In addition, fecundity of multiple-infected females affects the survival success of wOri in a similar way. Both the multiple-infected and double-infected males can suppress the fecundity of uninfected females. Although the multiple-infected males are stronger than double-infected males in depressing the fecundity of uninfected females, it is uncertain whether multiple-infection can succeed in the competition with double-infection.Several strains of et alWolbachia invasion and accelerate the rate of Wolbachia invasion in the super-infected A. albopictus population. In contrast, a higher threshold infection frequency and slower replacement rate is shown in the single-infected A. albopictus population, since it is driven only by CI. In addition, the rapid spread of Rickettsia sp. nr. bellii in a population of an invasive agricultural pest, the sweet potato whitefly, Bemisia tabaci is driven by fitness benefits and female bias wOri can be driven by the combination of CI shared with wCon and Cardinium, host longevity increase and attenuating effects on fecundity and longevity of uninfected females.Several studies have proved that the spread of bacterial symbionts can be driven by various host fitness benefits. Dobson Wolbachia. The new forces we found may provide insights into the distribution of Wolbachia bacteria among species. In addition, the new associations of inherited wOri and hosts that have been regarded as forces driving the spread of the symbiont may result in rapid evolution of both partners and phenotypic shifts that optimize the symbiosis WolbachiaIn conclusion, we describe novel forces driving the spread of


Salmonella with sensitivity of 500 cfu mL−1. With two monoclonal antibodies, which recognize different antigenic determinant on the surface of Salmonella, we prepared antibody-coated magnetic nanoparticles (MNPs) and conjugates of QDs-Ab. The immune-magnetic beads were verified with high enrichment efficiency for Salmonella (90%). A sandwich structure formed if the Salmonella solution was mixed together with immune-beads and QDs-Ab, and the fluorescent single from QDs was related to the amount of Salmonella. A linear response between fluorescence intensity and various concentrations of Salmonella (2.5 × 103 to 1.95 × 108 cfu mL−1) were observed with this proposed method. The total assay time for Salmonella was 30 min, and no cross-reaction to other microbial strains, such as Staphylococcus aureus, Escherichia coli (E. coli) and Escherichia coli O157:H7 (E. coli O157:H7), were found using this detection system. All our results showed that the simple homogeneous immunoassay could be applied in Salmonella screening without time-consuming extra-enrichment of bacteria.Simple immuno-magnetic separation tandem fluorescent probes based on quantum dots-antibody (QDs-Ab) were developed to detect  Salmonella is a common food-borne pathogen that causes food contamination, which has resulted in higher economic losses and poses a significant threat to public health [Salmonella infections have been increasing in recent decades. The most easily contaminated foods by Salmonella are meat; poultry and egg products [Salmonella screening method is crucial to safeguard human health and identify their distribution for better management of food safety.c health ,2. Salmoc health . These ic health –6. Annuac health ,8. Even products . Hence, Salmonella detection have been developed, including conventional culture methods, immunoassays [To date, some approaches for noassays –17 and pnoassays –22. HoweSalmonella was used in our detection design. One antibody was conjugated to QDs for detection and the other was bound to MNPs for capturing the targeted bacteria in solution. A fluoresce single was quantitative based on the forming a sandwich structure (QDs-bacteria-MNPs).In this paper, we present a novel immuno-sensor using quantum dots (QDs) as fluorescent label and magnetic nanoparticles (MNPs) as enrichment reagent. A pair of monoclonal antibody against Salmonella, Staphylococcus aureus, E. coli and E. coli O157:H7. The paired monoclonal antibodies, QDs and MNPs were all prepared in our lab. Two antibodies were obtained through hybridoma selection process. The affinity constants were calculated as 1.8 × 109 and 2.2 × 109, respectively, for these two anti- Salmonella antibody.All bacterial strains used in this study were purchased from the China Center of Industrial Culture Collection , including N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) was purchased from J&K Scientific, Ltd. . 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was purchased from Aladdin Reagent . Biotin and streptavidin were purchased from Sigma-Aldrich . Other chemicals were purchased from the Shanghai Chemical Reagent Company . All water in this experiment was ultra-water from Millipore water purification system .Bovine serum albumin (BSA) was obtained from Solarbio Science & Technology, Co, Ltd. . Salmonella antibodies, were used in this detection system. The anti-Salmonella antibodies were immobilized individually on the MNPs and QDs. The two antibodies recognize different antigenic determinants of Salmonella. For assay, the sample solution was sufficiently incubated with immune-beads and QD probes in a tube for a period. Then, the tube was put in a magnetic field for separating the MNPs-bacteria-QDs from solution. The isolated Salmonella coupled with the QDs was quantified using a fluorescence spectrophotometer . The increasing Salmonella concentrations allow more QDs to be captured by magnetic beads resulting in more intensive fluorescence single.A schematic of the sensing principle was illustrated in −1, respectively. One milliliter of the linker solution was mixed with equal volume of MNPs and held for 15 min at room temperature, then 100 μL aliquots of anti-Salmonella antibody (Ab-1) was added drop-wise into the solution. The reaction was carried out at 37 °C for 2 h, and then the MNP beads were isolated using magnet. After removal of the liquid in the tube, 1% BSA solution in PBS  was re-dispersed on MNPs with continuous stirring for 1 h at 37 °C to block the bareness sites on the surface of MNPs. Following another magnetic separation step, the residues were dissolved in 1 mL PBS and then stored at 4 °C for further use. The capture ability of Salmonella from immune-magnetic beads was evaluated using the counted Salmonella (1.16 × 105 cfu mL−1). After half an hour’s incubation, the immune-magnetic beads were recovered using magnetic separation.The MNPs used here was modified with carboxyl group on their surface (particle size 100 nm). EDC and sulfo-NHS were applied as linkers in this experiment and they were dissolved to phosphate-buffered saline  with final concentrations of 0.5 and 0.2 mg mLThe separated MNPs were washed 5 times and re-suspended in nutrient broth. Then, plate counts were performed to evaluate the enrichment efficiency of immune-magnetic beads.Salmonella antibody (Ab-2) and QDs were coupled by a biotin-streptavidin bridge. The conjugation method is the same as our previous reports [The anti- reports ,24. The Salmonella/QDs-Ab. With a magnetic separation, the residual QDs were removed from the solution. The Salmonella cells bound to the QDs and MNPs were re-suspended in 1 mL PBS for fluorescent measurement. Various concentrations (from 102 to 108 cfu/mL) of Salmonella in PBS buffer were tested with this proposed detection system. The values of the fluorescence intensity were plotted against the Salmonella to produce a linear curve. The specificity of this proposed immunoassay was tested with other bacteria, including Staphylococcus aureus, E. coli and E. coli O157:H7.The sample solution (1 mL) was added with 50 μL of immune-MNPs beads, and following this, 50 μL of QDs-Ab conjugates were added. The mixture solution was allowed for 30 min incubation forming the sandwich structure of immune-MNPs/4 reduction beforehand [Both antibody and QD were functioned using biotin, and then two parts were bridged using streptavidin through the specific affinity reaction. The successful preparation of Ab-biotin complex was verified with UV detection. As it was shown in forehand . The UV With the connecting agent of streptavidin, the antibody was successfully anchored on the surface of QDs. The original QD showed the typical fluorescence emission peak at 620 nm, while an obvious red shift of the conjugates of QD-dBSA-biotin  and QD-Ab (630 nm) were seen in Salmonella by immune beads was calculated as (1.05 ± 0.12) × 105 out of the total amount of 1.15 × 105 cfu mL−1, resulting in the average 90% binding efficiency.With gradient dilutions and then plate counts, the absorbed Salmonella was 1.16 × 105 cfu for optimization tests, and a blank sample  was used as a control. The photoluminescence (PL) intensity from the formed sandwich products was determined. The results from Salmonella.In evaluation of the optimum incubation period for sample solution, immune-beads and QD probes in the assay buffer, various incubation times  were tested. The total amount of Salmonella concentrations (500 to 1.95 × 108 cfu mL−1). As shown in Salmonella. It demonstrated a good linear relationship when the bacteria concentration ranging from 2.5 × 103 to 1.95 × 108 cfu mL−1 with a correlation coefficient of 0.9936 . All the results indicated that the immune-MNPs tandem QDs-Ab system was very specific in Salmonella detection.In specificity tests, the control sample (no containing any bacteria) and three pathogens, including Salmonella here. With the efficient enrichment of immune-MNPs, the sample could be completed in half an hour. Due to the predominant fluorescent character of QDs, quantitative analysis was possible for Salmonella with a linear curve. Without complex pre-culturing procedures, the proposed method could be directly detected samples with the sensitivity of 500 cfu mL−1 for Salmonella. This immunoassay was simple and fast, which makes it a great potential for being applied in surveillance of Salmonella contamination.A sensitive and simple immunoassay was developed for


Increased use of the Internet provides new opportunities for collecting data in large studies. The aim of our new Web-based questionnaire, Active-Q, is to assess total physical activity and inactivity in adults. Active-Q assesses habitual activity during the past year via questions in four different domains: (1) daily occupation, (2) transportation to and from daily occupation, (3) leisure time activities, and (4) sporting activities.The objective of our study is to validate Active-Q’s energy expenditure estimates using the doubly labeled water (DLW) method, and to assess the reproducibility of Active-Q by comparing the results of the questionnaire completed by the same group on two occasions.The validity and reproducibility of Active-Q were assessed in a group of 37 individuals, aged 20 to 65 years. Active-Q was distributed via email to the participants. The total energy expenditure of the participants was assessed using DLW for 11 consecutive days.The median time to complete Active-Q was 6.1 minutes. The majority of participants  reported that the questionnaire was “easy” or “very easy” to answer. On average, Active-Q overestimated the total daily energy expenditure by 440 kJ compared with the DLW. The Spearman correlation between the two methods was r = 0.52 (P < .001). The intraclass correlation coefficient for total energy expenditure between the results of Active-Q completed on two occasions was 0.83 (95% CI 0.73-0.93).Active-Q is a valid and reproducible method of assessing total energy expenditure. It is also a user-friendly method and suitable for Web-based data collection in large epidemiological studies. Increased use of the Internet over the past decade provides new opportunities to use Web-based questionnaires in epidemiological studies. Compared to traditional paper-based questionnaires, Web-based alternatives may simplify data collection and improve the quality of data -3. The nAt present, there are few physical activity questionnaires available specifically developed for the Web. To the best of our knowledge, only one Web-based questionnaire  has been validated and showed acceptable results . TherefoThe primary aim of this study was to test the validity of Active-Q against the doubly labeled water (DLW) method, the criterion standard for measuring energy expenditure . FurtherStudy participants of both sexes, aged 20 to 65 years, were recruited in April of 2009 through public advertisements (including advertisements on the campuses of three universities) around Stockholm, Sweden. Participants were required to have an email address and access to the Internet. Exclusion criteria were any form of weight alteration diet, pregnancy, or having given birth during the ten months prior to the start of the study. Participants were provided with written and verbal information about the study. All participants gave their written informed consent prior to entering the study.In total, 40 individuals were recruited. Data from three participants were excluded from the analysis because of illness during data collection, incomplete data from the first Active-Q questionnaire , and unreliable DLW data, respectively. After exclusions, data from 37 participants remained for analysis.The study was approved by the Research Ethics Committee at the Karolinska Institutet, Stockholm, Sweden.Active-Q is a Web-based questionnaire designed to assess physical activity and inactivity in adults older than 18 years. Respondents are asked to report their habitual activity during the past year. Active-Q covers four different domains: (1) daily occupation, (2) transportation to and from daily occupation, (3) leisure time activities, and (4) regular sporting activities see . The leiIn total, Active-Q includes 35 questions with all activities linked to a corresponding Metabolic Equivalent Task (MET) value , 9. Tabl-1 ∙ h-1 [Energy expenditure (EE) was estimated based on the assumption that 1 MET equals 1 kcal ∙ kg-1 ∙ h-1 . To calcactivity (kJ/day) = METactivity ∙ Weight (kg) ∙ Durationactivity (h/d) ∙ 4.184EETotal energy expenditure was expressed in terms of the crude total energy expenditure and the total energy expenditure per 24 hours. The crude total energy expenditure was obtained by summing the contributions of energy from each activity in Active-Q. The total energy expenditure over a 24-hour period was calculated by adding eight hours of sleep to the crude results from Active-Q. If the resulting time differed from 24 hours, time was added or subtracted to obtain the adjusted total energy expenditure per 24 hours. A MET value of 2.0 was assumed for the time added and subtracted , 10.2H2O  to 1 L 10% normalized H218O . Participants gave a baseline urine sample before drinking either 108 g (participants < 75 kg) or 141 g (participants ≥ 75 kg) of the bulk dose depending on their self-reported weight at the beginning of the study. In addition, participants collected 5 ml daily urine samples (excluding the first morning void) on each of the following 10 days. Participants recorded the date and time of each sample collection and kept the samples refrigerated until they were returned to the research team. The research team sent the samples to MRC Human Nutrition Research in Cambridge, UK, for analysis.Doubly labeled water was used as the criterion measure of total energy expenditure  for 11 c2H analysis, 0.4 ml samples of undistilled urine were equilibrated with hydrogen gas using platinum catalyst rods to promote the rapid exchange between2H and1H. The 3.5-ml sample vials were first flush-filled with hydrogen, producing approximately 3 ml of gas at 1 bar atmosphere, and then equilibrated at 22.0 +0.1°C for 6 hours. Cryogenically dried hydrogen gas from the headspaces of each sample was analyzed using isotope ratio mass spectrometry .The principles for the slightly modified analyses of the isotopic enrichment in the samples have been described in detail in previous studies , 13. Bri18O/16O ratios were made using an AP2003 continuous-flow isotope ratio mass spectrometer . Urine samples of 0.5 ml were placed in 10 ml Vacutainers , flush-filled with 5% CO2 in nitrogen and then equilibrated on blood tube rotators overnight at room temperature before analysis. In all cases, analytical standards prepared in-house and traceable to the international standards, Vienna Standard Mean Ocean Water (VSMOW) and Standard Light Arctic Precipitation (SLAP), were included in each batch of samples analyzed.Measurements of theTotal energy expenditure was calculated as described by Schoeller et al  from thet tests were used to assess potential differences. The level of significance was set at P < .05.Descriptive statistics were used to present the characteristics of the participants. Results are reported as mean values and standard deviations (SD), with the exception of time to respond to the questionnaire, which is presented as the median response time, and results of user-friendliness, which is presented in absolute numbers. For categorical variables or variables with a skewed distribution, Fisher’s exact test was performed to assess potential differences between men and women, and participants < 30 and ≥ 30 years of age. For those continuous variables typically normally distributed , The degree of association between the total energy expenditure obtained from Active-Q-I, both crude and adjusted to reflect a 24-hour day, and the DLW method, was assessed using Spearman correlation coefficients. Because Spearman correlation coefficients do not detect systematic differences between the methods, we used the Bland-Altman technique  to deter2. Men were taller (P < .001) and weighed more (P < .001) than women. Subjects ≥ 30 years of age, weighed more (P = .024) and had a higher BMI (P = .007) compared with participants < 30 years of age. No other statistically significant differences were found between sex or age groups.P = .006). No other statistically significant age-related or sex-related differences were seen in the results.The median time required to fill out the Active-Q questionnaire on the first occasion was 6 minutes and 6 seconds. On average, the activities reported corresponded to 11 hours and 24 minutes of a typical day (excluding time spent sleeping). In the evaluation of the user-friendliness of the questionnaire, the majority of respondents  graded the questionnaire as “easy” or “very easy” to answer. Respondents were also asked to give the questionnaire an overall grade on a scale from 1 (worst) to 5 (best). The majority of respondents  graded the questionnaire a 4. An equal number of participants , graded the questionnaire a 3 or a 5. The remainder of participants  gave the questionnaire a 2. No respondents gave the questionnaire a 1, the worst possible grade. The mean overall grade for Active-Q-I was 3.9 ±0.8. Participants ≥ 30 years graded the questionnaire higher than younger participants , while the mean energy expenditure from Active-Q-I and Active-Q-II, adjusted to reflect a 24-hour period, was 11,667 kJ (SD 3212) and 11,529 kJ (SD 2758), respectively. The mean crude energy expenditure assessed with Active-Q-I was 7008 kJ (SD 3854). The crude energy expenditure for each domain of Active-Q-I was 2971 kJ (SD 1736) for daily occupation, 434 kJ (SD 388) for transportation, 2243 kJ (SD 1550) for leisure time activities, and 1360 kJ (SD 3044) for regular sporting activities. The mean crude energy expenditure from Active-Q-II was 6439 kJ (SD 2614). The crude energy expenditure for each domain of Active-Q-II was 3005 kJ (SD 1537) for daily occupation, 365 kJ (SD 296) for transportation, 2254 kJ (SD 1880) for leisure time activities, and 815 kJ (SD 761) for regular sporting activities.r = 0.42 (P = .01). After adjusting Active-Q-I for a 24-hour day, the Spearman correlation coefficient increased to r = 0.52 (P < .001).The Spearman correlation coefficient between the crude total daily energy expenditure assessed with Active-Q-I and DLW was 2.The intraclass correlation coefficient for the crude total energy expenditure assessed from the questionnaire on two occasions was 0.66 (95% CI 0.47-0.84). After adjustments to a 24-hour day, the value increased to 0.83 (95% CI 0.73-0.93).The results of this study demonstrate that Active-Q is a user-friendly questionnaire that provides valid and reproducible estimates of total energy expenditure when compared with objective DLW measurements, the criterion standard .The validity and reproducibility of many paper-based physical activity questionnaires in use today are low , althougImportant factors in making a questionnaire user-friendly are the length and the level of details in a questionnaire, plus the way in which questions are structured and the order in which they are presented . It is aToday, more than 90% of the population in Sweden between 16 and 74 years of age have access to the Internet at home . TherefoAmong Swedish individuals between 16 and 44 years of age, over 97% reported to be frequent users of the Internet compared to 90% for individuals between 45 and 54 years, and 70% for individuals between 55 and 74 years . AlthougIdeally, in validation studies, the reference method should reflect the same period of time as the new method being validated. However, habitual physical activity was assessed over a one-year period with Active-Q, while that measured with the DLW method reflects physical activity over 11 days. Repeated reference measures of DLW over a longer period of time would have been preferable, but this was neither practical nor possible in our study setting. Another drawback of this study is that we were only able to validate measures of total energy expenditure. Because the DLW method does not discriminate between different levels of intensity in activity, we were not able to validate Active-Q in this respect. Since different aspects of activity may affect health-related outcomes differently, the ability of Active-Q to accurately assess this needs to be validated in future studies using, for example, accelerometers.When estimating the total daily energy expenditure with Active-Q, we assumed that all participants slept for eight hours . The truThe use of published MET values  to repreWhile the point estimate of the intraclass correlation for the adjusted total energy expenditure assessed with Active-Q is high, the point estimate for the assessed crude total energy expenditure is lower, and the confidence intervals around both estimates are rather wide. The wide confidence intervals, and the rather low point estimate obtained for the crude total energy expenditure, might be explained by our small sample size and sampling variability. To obtain more certain measures of reproducibility, we need to conduct a larger study to assess reproducibility of both assessments of total energy expenditure, as well as energy expenditure within each individual domain of Active-Q.Our study population was recruited from the Stockholm area, including the campuses of three universities, and the participants were young and predominantly female. This may limit the generalizability of our results. The overall level of education among the participants was high, with more than 80% having an education longer than 12 years, compared with 36% in the general Swedish population aged 16-64 years . Higher To the best of our knowledge, Active-Q is one of the first validated physical activity questionnaires specifically designed for Web-based use. Despite the limitations mentioned previously, we believe Active-Q to be a good assessment method in studies collecting Web-based data. Active-Q is currently in use in three large ongoing cohort studies in Sweden, aimed at recruiting hundreds of thousands of people .An increasing number of studies are relying on the collection of data via the Web, and there is a need for valid Web-based methods of assessing physical activity. We have demonstrated that Active-Q is a valid method for estimating total energy expenditure. Furthermore, Active-Q is also a reproducible and user-friendly method. We conclude that Active-Q is a suitable method for collecting Web-based data on physical activity and inactivity in large epidemiological studies.


Peony is often used in Chinese herbal medicine for the treatment of depression-like disorders. Our previous studies have demonstrated that the total glycosides of peony exert antidepressant-like effects in animal models. Paeoniflorin is the main active glycoside of peony. The aim of this study was to evaluate the antidepressant-like effects of paeoniflorin in mice, as well as its active mechanisms. The results revealed that intraperitoneally injected paeoniflorin significantly reduced the duration of immobility in forced swimming and tail suspension tests. The doses that affected the immobility response did not affect locomotor activity. Furthermore, paeoniflorin antagonized reserpine-induced ptosis, akinesia and hypothermia. Paeoniflorin also significantly increased the levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus. These results suggest that the upregulation of serotonergic systems may be an important mechanism for the antidepressant-like effects of paeoniflorin in mice. Paeonia lactiflora Pall. (Ranunculaceae), commonly known as peony, is a commonly used in herbal medicines in China, Korea and Japan. It is a component herb of numerous traditional formulae, including Jiaweisinisan and Dang Gui Shao Yao San, prescribed for the treatment of depression-like disorders  reuptake inhibitors, are widely available in the pharmaceutical market. However, the majority of these antidepressants have undesirable side-effects -3. Conseisorders ,5. The aisorders . Paeonifisorders -13. Howeisorders . In the ad libitum and were left to acclimate for 4 days before use in experiments. The use of experimental mice was approved by the Animal Experimentation Ethics Committee of the Zhejiang Chinese Medicine University and conducted in accordance with the National Institutes of Health (NIH) Principles of Laboratory Animal Care .Male Institute of Cancer Research (ICR) mice (18–22 g) were obtained from the Laboratory Animal Unit of Zhejiang Chinese Medicine University . The animals were housed five per cage and acclimatized to a colony room with controlled ambient temperature (24±1°C), humidity (50±10%) and a 12 h light/dark cycle. They were fed a standard diet and water Paeoniflorin (purity >98%) was purchased from Guizhou Dida Technology Co., Ltd. . Imipramine hydrochloride was purchased from Sigma-Aldrich  as a positive control. Reserpine was purchased from Bangmin  and 5-hydroxytryptamine (5-HT), NA, dopamine (DA) and 5-hydroxyindoleacetic acid (5-HIAA) were purchased from Sigma-Aldrich. For intraperitoneal injection, paeoniflorin, imipramine and reserpine were separately dissolved in 0.9% normal saline and diluted to the desired concentration on the day of testing. In this study, various doses of paeoniflorin  and imipramine (10 mg/kg) were injected intraperitoneally (i.p.) 60 min before testing.et al ]. The mixture was stored on ice for 1 h and then centrifuged at 12,000 × g (4°C) for 20 min. Then, 20 μl of the resultant supernatant was directly injected into an ESA liquid chromatography system equipped with a reversed-phase C18 column  and an electrochemical detector . The detector was set at 450 mV. The mobile phase consisted of 125 mM citric acid-sodium citrate (pH 4.3), 0.1 mM EDTA, 1.2 mM sodium octanesulfonate and 16% methanol. The flow rate was 1.0 ml/min. NA, 5-HT, DA and 5-HIAA were identified and quantified by comparing their retention times and peak areas to those of standard solutions. The contents of 5-HT, NA, DA and 5-HIAA were expressed as ng/g in wet weight tissue.Levels of 5-HT, NA, DA and 5-HIAA in the hippocampus were measured by high-performance liquid chromatography (HPLC) coupled with an electrochemical detector as described previously . BrieflyThe results were expressed as the mean ± standard error of the mean. All data were analyzed statistically using one-way analysis of variance, followed by Dunnett’s test. P<0.05 was considered to indicate a statistically significant difference.Paeniflorin at doses of 20 and 40 mg/kg significantly decreased the duration of immobility in the tail suspension test compared with the control group (P<0.05). Imipramine (10 mg/kg) treatment also had a significant effect on immobility P<0.01; .Paeoniflorin (40 mg/kg) significantly inhibited immobility in the forced swimming test (P<0.05). Imipramine (10 mg/kg) treatment also decreased the duration of immobility P<0.01; .The effect of paeoniflorin on the locomotor activity of mice is shown in The effects of paeoniflorin on reserpine-induced ptosis, akinesia and hypothermia are shown in The effect of paeoniflorin on hippocampal monoamine neurotransmitters and a major metabolite of 5-HT in mice is presented in The tail suspension and forced swimming tests are widely used for the screening of antidepressant activity ,18. The In these behavioural tests, false-positive results are occasionally obtained for agents that stimulate locomotor activity . TherefoReserpine is an antihypertensive drug that depletes neuronal storage granules of biogenic amines in the brains of rodents and produces a clinically significant depression-like state . Mice beIn a conclusion, upregulation of serotonergic systems may be an important mechanism in the antidepressant-like effects of paeoniflorin in mice.


Amphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals., Lithobates pipiens  with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions.The method is based on simultaneous injection of both female and male leopard frogsL. pipiens in the breeding season. The combination of des-Gly10, D-Ala6, Pro-NHEt9-GnRH  with metoclopramide hydrochloride  or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5–6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully.We tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.We provide evidence for successful captive breeding of the leopard frog,  Lithobates pipiens (formerly Rana pipiens) in the U.S.A. were already declining in the mid-1960s. Today, Western North American populations, including those in Alberta, British Columbia, Colorado, and Nevada have dramatically declined [L. pipiens have also been declining over the last 40 years [Global amphibian loss is considered a major factor in the contemporary "sixth mass extinction" according to Wake and Vreedenberg . The potdeclined -10. Nort40 years .Nace and colleagues ,12 were Rana temporaria[Rana ridibunda indicates that DA can be delivered to the pituitary [R. temporaria in some situations [R. temporaria[Bufo fowlerii. In that study, they used the DA antagonist pimozide and concluded that pimozide may increase spawning in some situations and hormone combinations. However, pimozide is not specific to DA receptors, and acts on adrenergic and serotoninergic receptors in addition to DA receptors [L. pipiens.While it is now known that GnRH and GnRH agonist treatments can stimulate pituitary luteinizing hormone (LH) release in frogs ,15, theyemporaria increaseemporaria and DA temporaria,22. Immuituitary . The DA tuations . Moreoveemporaria. These demporaria. Browne emporaria exploredeceptors , so it’sL. pipiens, it was clear that the combination of des-Gly10, D-Ala6, Pro-NHEt9-GnRH (GnRH-A) and the specfic DA D2-receptor antagonist MET gave the best results. We named the approach the AMPHIPLEX method, a term that derives from the combination of the words amphibian and amplexus. Amplexus refers to the specific reproductive behaviour of frogs where the male grasps the female, helping to stimulate ovulation and after some delay fertilizes the eggs as they are laid. In one year in the breeding season, we obtained fertilized egg masses from 100% of females, while in the second year this was only 60% [L. pipiens a good first step [L. pipiens[In our first report on hormonal induction of spawning in only 60% . We consrst step . On the . pipiens, indicatL. pipiens following co-injection of GnRH-A and either of the DA antagonists MET or domperidone. Given the efficacy and ease of use of the GnRH-A+MET combination, we also successfully induced breeding out-of season and obtained thousands of viable tadpoles. In our case, the main reason for this planned breeding is to obtain healthy tadpoles in a timed manner for physiological, ecotoxicological and epidemiological studies [L. pipiens is unfortunately in decline in several regions of the traditional range [Here we report on the efficient, large-scale spawning induction during the reproductive season of  studies ,29. In tal range -12.Mature leopard frogs  were collected near Bishops Mills, Ontario during their spring migration to breeding pools between April 9 and 13, 2011. The animals were kept cold (4°C) temporarily and were transported on April 19 (water temperature 6-7°C) to the National Wildlife Research Centre, Ottawa and housed outside in large 380 L high-density polyethylene (Rubbermaid) tanks containing 200 L of water as described previously . The sex10, D-Ala6, Pro-NHEt9-GnRH ; GnRH-A) and metoclopramide  as reported previously [Animals were injected between 12:00–17:00 h on April 30, 2011. Animals in the control group  were injected intraperitoneally (i.p.) with saline  and DMSO (1 μl/g) vehicles using a 26-gauge needle attached to a disposable 1-ml syringe. The AMPHIPLEX method is based on injections of a mixture of des-GlyOn Day 5 post-injection, the resulting egg masses were weighed and placed in individual aquaria at room temperature (21°C) to observe and record embryonic development. Post-spawning body weight of females was also recorded. Additionally, 3 small subsamples of the egg masses were weighed and the number of eggs counted under a dissection microscope. The average number of eggs per gram of subsample was multiplied by total egg mass weight to estimate total fecundity. Relative fecundity was estimated by dividing the total number of eggs by the pre-spawning body weight of females. Percent fertilization rates were estimated by determining the proportion of developing and non-developing embryos 4 days following spawning.It should be noted that the females that did not spawn were sacrificed and it was determined that they did have eggs.For the breeding trials that occurred outside the normal spring breeding season, mature leopard frogs were collected near Bishops Mills, Ontario from mid November-early December 2011 using submerged minnow traps in a deep pond, which is a traditional, successful hibernation site for leopard frogs in this region. The animals were kept cold (4°C) temporarily and were transported and housed in-doors in tanks at 4°C at the University of Ottawa aquatic facility. Mean (± SD) body weights for all females and males used in this experiment were respectively 51.9 ± 8.5 and 25.5 ± 6.6 g. Two trials were conducted.In the first trial, 7 females and 7 males were placed separately in breeding tanks  and maintained at 4-6°C, in the dark for 23 days. Thereafter they were exposed to 6h light  for one day, then 14h light  and maintained at 4-6°C, in the dark for 5–8 weeks. Thereafter, they were exposed to the same photoperiod/temperature regimen described above for Trial 1. For the priming pre-treatment, females and males were injected i.p. between 14:00–14:30 with a low dose of GnRH-A . Animals were moved to the breeding tanks but the sexes were kept separated. Twenty-four hours later (Day 2), animals were injected i.p. with GnRH-A+MET. Immediately following this injection, 3 females and 4 males were placed in a given breeding tank (16-17°C).For both trials in Experiment 2, the resulting egg masses were weighed and the post-spawning body weight recorded. Fertilization and fecundity measurements followed the protocol described for Experiment 1.The females that did not spawn were sacrificed and it was determined that they did have eggs.Three egg masses obtained from pairs injected only with GnRH-A+MET (masses 1–3) and 2 egg masses from the GnRH-A priming protocol (masses 4–5) were further assessed. These 5 egg masses were studied to determine tadpole survival, growth, and metamorphic rates in addition to potential genetic  influences on these parameters. Five fertilized egg masses were removed from the breeding tanks from the out-of-season breeding trials reported above and placed individually in 20 L glass tanks at room temperature until hatching and development to Gosner Stage (Gs) 25. . PercentAt approximately Gs40, a floating Styrofoam plate and plastic grill was placed in each tank so that tadpoles could rest, and prepare to leave the water at metamorphosis. To reduce the chance of drowning, upon reaching Gs45 froglets were transferred to 100 × 15 mm glass Petri dishes with 5 ml water. Once metamorphosis was completed , animals were anaesthetised in tricaine-methanesulfate  and sacrificed.3)*100. Survival at Gs45 was calculated by comparing the number of tadpoles reaching that stage with the total number of Gs25 embryos at the beginning of the experiment. The percentage of Gs25 embryos that completed metamorphosis (Gs46) was also determined.For each metamorph, the snout-vent length , weight , type and incidence of deformities, and total days post-fertilization to complete metamorphosis (DTM) were recorded. The condition factor (k) was calculated using SVL and weight measurements as follows: k = (W(g)/SVL(cmhttp://www.graphpad.com) and Student’s T-test (http://studentsttest.com) were used. Data are reported as mean ± SD.Online freeware was used for statistical analysis of spawning induction data. Fisher’s Exact test . Normality was verified using Shapiro–Wilks’ test and logarithmic transformations were performed to ensure normality when required. Homoscedasticity of variances was verified with Levene’s test. A one-way analysis of variance (ANOVA) was used to test for statistical differences between egg masses in SVL, W, K and DTM. Differences in percentage of animals that complete metamorphosis, incidence of malformations, survival to Gs45 and death rate between Gs45 and Gs46 were determined using the non-parametric test Krustal-Wallis. Data are expressed as mean ± SD. In all tests, differences were accepted as significant when p < 0.05.All procedures followed the animal care guidelines of the University of Ottawa and the Canadian Council on Animal Care.One pair of saline-injected control animals was observed in amplexus on Day 3 This couple remained in amplexus for 2 days but did not spawn.In the treated groups, the first egg masses were seen on Day 3. On Day 3, 5/18 pairs were in amplexus and 2 egg masses were laid in the GnRH-A+MET group. On Day 3, 9/17 pairs were in amplexus and one egg mass was laid in the GnRH-A+DOM group. By Day 5, spawning activity stopped. Regardless of treatment, all egg masses laid were fertilized (>90%) and led to development of healthy tadpoles. In the GnRH-A+MET group 16/18 (89%) females laid eggs. In the GnRH-A+DOM group 15/17 (88%) females laid eggs  in the GnRH-A+MET group and 20.3% (range 10.2-35.1%) in GnRH-A+DOM group . Total egg mass weight was also not different  between the treatment groups and was 137.4 ± 34.5 g for GnRH-A+MET and 130.3 ± 27.2 g for GnRH-A+DOM. Similarly, the total number of eggs per egg mass were not different  between the treatments and were 2764 ± 690 and 2642 ± 597 for GnRH-A+MET and GnRH-A+DOM, respectively . The total number of eggs per egg mass was 2,052 ± 871 in Trial 1 and 1,584 ± 777 in Trial 2 . Relative fecundity as indicated by eggs per gram pre-spawning body weight was estimated as 36 ± 15 and 31 ± 14 for Trial 1 and Trial 2, respectively . We recorded high % fertilization levels in Trial 2 of Experiment 2. On average this was 85% (range 53-98%) and was significantly higher  than fertilization estimates obtained in Trial 1 , there r female . It willAnother important consideration addressed in our study is the development of tadpoles obtained from captive breeding outside of the normal reproductive period. Generation of tadpoles for physiological and ecotoxicological experiments, and for captive breeding and reintroduction programs of endangered species all require large numbers of normal healthy tadpoles that grow and metamorphose. An additional requirement for reintroduction programs is the maintenance of genetic diversity, and therefore, careful management of broodstock and their offspring. We studied the 3 egg masses obtained from pairs injected only with GnRH-A+MET (masses 1–3) and 2 egg masses obtained from the GnRH-A priming protocol (masses 4–5). Levels of fertilization varied from 10–97.2%, but the variations in developmental parameters measured were not related to this fertility estimate. The number of days from fertilization to metamorphosis (DTM) varied from 85–118, which is well within the wide range 7–42 weeks) found in the literature ,39-41. S weeks foL. pipiens. This species may serve as a good model to use for the further development of robust spawning induction methods for endangered anurans because until now it has been considered very difficult to breed in captivity. The AMPHIPLEX method to inject both sexes simultaneously with a solution containing a GnRH agonist and a DA antagonist has been used successfully in 6 anuran species [We provide evidence for successful captive breeding of the leopard frog,  species ,43,44.The authors declare that they have no competing interests.L. pipiens and wrote the paper. FS collected all L. pipiens and contributed to development of spawning conditions required. LNM and CKM designed and conducted the developmental studies. EB and BF contributed to the feeding, maintenance and hibernation protocols. AB performed some of the fertility studies. LT contributed to the design of the out-of-season breeding and tadpole maintenance protocols. All authors read and approved the final manuscript.VLT developed the spawning induction method, designed, performed all breeding experiments with Lithobates pipiens by Dr. A. Morin .Tadpoles and froglets resulting from induced spawning out of the normal breeding season using the AMPHIPLEX method. Shown are photographs of Click here for file


A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both .Malyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic COC. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes.Here we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures , their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for malate synthases. However, a comparison of the two MCL structures with known malate synthases raised the question why the MCLs are not also able to hydrolyze CoA thioester bonds. Our results suggest the previously proposed reaction mechanism for malate synthases may be incomplete or not entirely correct. Further studies involving site-directed mutagenesis based on these structures may be required to solve this puzzling question.Although the  The data was processed with XDS  and the DB 1U5H)  as the sDB 1U5H) . Furtherhttp://blast.ncbi.nlm.nih.gov/]. Accession numbers for reference sequences are provided in Table -60 was applied for all BLAST searches except for those using the β-subunit of citrate lyase of Klebsiella pneumonia as query in which the cut-off was 1e-80. All sequences that appeared to be truncated were removed from the data set. All sequences that were more than 90% identical to another one were removed with CD-HIT [E. coli malate synthases A and G  were added and alignment was performed with the BioEdit program [http://www.mbio.ncsu.edu/bioedit/bioedit.html] using the similarity matrix PAM250. The phylogenetic tree was constructed from a total of 675 amino acid sequences using MEGA5 [Amino acid sequence searches within the domains of Bacteria and Archaea were performed using BLAST [h CD-HIT  to furthng MEGA5  and the ng MEGA5 . A bootsng MEGA5  was infehttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html]. Protein-protein interactions were also analyzed by use the PIC-webserver tool [http://pic.mbu.iisc.ernet.in]. Hidden Markov models (HMM) and HMM logos were created using HMMER Version 3.0 [Chimera  was usedver tool  [http://sion 3.0  and Logosion 3.0 , respectsion 3.0 .The authors declare that they have no competing interest.JZ and CAK designed the research. JZ produced and crystallized the enzymes, collected and processed the diffraction data, and solved, modeled and refined the structures. JZ and CAK analyzed the structures. JZ and CAK wrote the paper. Both authors read and approved the final manuscript.2+ ion are labeled. The region responsible for the movement of the C-terminal lid domain is marked “bending”. Secondary structure elements derived from a PDBsum analysis of the MCLC structure (PDB 4L80) are aligned with the HMM-logo.HMM–Logo of the amino acid sequences of enzymes that cluster together with MCLC in the phylogenetic tree. Numbering of residues corresponds to MCLC. The 48 sequences comprised in this figure share at least 57% sequence identity. Catalytic residues are marked with asterisks. Residues that coordinate the MgClick here for file2+ ion are labeled. The region responsible for the movement of the C-terminal lid domain is marked “bending”. Secondary structure elements derived from a PDBsum analysis of the MCLR structure (PDB 4L9Z) are aligned with the HMM-logo.HMM-Logo of the amino acid sequences of enzymes that cluster together with MCLR in the phylogenetic tree. The numbering of residues corresponds to MCLR. Only sequences were used  that share at least 50% sequence identity to MCLR. Catalytic residues are marked with asterisks. Residues that coordinate the MgClick here for fileS)-citramalyl-CoA. The position of the β-carbon of citramalyl-CoA (formerly keto-carbon of pyruvate) is slightly shifted and its bonds assume a tetrahedral geometry compared to the planar geometry of pyruvate in A.Superpositions of Fo-Fc electron density simulated annealing omit maps on refined ligands for the malate synthase of H. volcanii. A) Omit map at 2.5 σ for acetyl-CoA and pyruvate. The α-carbon of the acetyl moiety is in very close proximity to pyruvate (2.4 Å). B) Omit map at 2.5 σ for (Click here for file